Document gEbGXR0En28kjdzjJ6pXqG969

ARNI6-0428 ATtotxaiccohlmoegnytsSttuodLieetstaenrdtoOCt.heArueIrnfdoartmeadtiMoan yon25P, F20O0A0 Genotoxicity 1) LAinneAsfsoraythoefTCeesltlCThreamniscfaolrmTa-t2io9n42anCdoCCy,toEtnovxiircointmyeinntatlhePCat3hHol1o0gTy L1a/2boCrlaotnoarly,CeSltlone: Research Laboratories, UniversityofMinnesota, 3M Ref. No. FC-143 Lot 340, L138679, March 5, 1981 2) FSianlamloRneeplolrat/,MMiuctraogseonmiecPlEavtaeluTaestti,onLiotftoTn-2B0io1n5etCicosC, in the Ames Inc., Study No. 20838, 3M Ref. No. FC-143, February 1978 3) CFionmalinRgepHoarzte,lMtuotna,gIennci.,ciSttyudTeysNto.on1T7-765506-04-i4n55a,n 3InMRievfo.MNoou.sFeCM-i1c0r1o5n,uLc1l3e1us67A,ssay, straight-chain APFO, November 1, 1996 4) Final Report, Mutagenicity Test Chinese Hamster Ovary (CHO) on T-6564 Cells: with MaeCaosnufriirnmgatCohrryoAmsossaoymwailthAbMeurlrtaitpiloens in HLa1r3v1e6s7t,s,sCtroamigihntg-chHaaiznelAtPoFnOI,ncS.,epStteumdbyeNro.161,71795906-0-437CO, 3M Ref. No. FC-1015, 5) FCionlail/RMeapmomrta,liMaunta-gMeincircoistoymTeesRtevweirtsheTM-u6t5a6t4ioinn tAhsesSaaylwmiotnhelalaCo-nEfsicrhmeartiocrhyiaAssay, Coming Hazelton Inc., Study No. 17750-0-409R, 3M Ref. No. FC-1015, L13167, straight-chain APFO, September 13,1996 6) `FiHnuamlaRnepWohrot,leMuBtlaogoedniLcyimtpyhToecsyttoens Tw-i6t3h4a2CMoenafsiurrmiatnogrCyhArsosmaoyswoimteh Aberrations Multiple in HLa1r3v3e6s4t,s,FC1o14m2i6n,gLHoatze1l, tsoond,iIunmc.p,eSrtfuludoyrNooo.cta1n7o0a7te3,-0N-o4v4e9mCb0e,r31M, Ref. 1996 No. FC-1090, 7) FCihnianlesReepHoartm,stMeurtaOgveanricyi(tCyHTOes)tCoenllTs:-6w3i4t2h aMeCaosnufriirnmgatCohrryoAmsossaoymwailthAbMeurlrtaitpiloens in HLa1r3v3e6s4t,s,FC11o4m2i6n,gLHoatze1,ltsoond,iIuncm.,peSrtfuludoyrNooo.cta1n7c0at7e3,-0S-e4p3t7eCmOb,er3M16,Re1f9.96No. FC-1090, 8) FCionmalinRgepHoratz,elMtuotna,gIennci.,ciSttyuTdeysNto.on1T7-06733-402-i4n55a,n 3InMVRievfo.MNoou.sFeCM-i1c0r9o0n,uLc1l3e3us64A,ssay, F11426, Lot 1, sodium perfluorooctanoate, December 14, 1995 9) FCionlail/RMeapmomrta,liMaunta-gMeincircoistoymTeesRtevweirtsheTM-u6t3a4t2ioinn AthsesaSya,lmCoonmelilnag-HEaszcehletroinc,hIinac., Study Npeor.fl1u7o0r3o0oc7t3a-n0o-a4t0e9,,D3ecMemRebfe.rNo1.4,F1C9-915090, L13364, F11426, Lot 1, sodium 00146 3 AN oN ICo tLe ASSAY OF CELL TRANSFORMATION AND CYTOTOXICITY IN C3H 10T 1/2 CLOMAL CELL LINE Submitted to the 3M Corporation st. ron eS, 34 Center sn : Dera59tintehentEronrvihrLoonbmeoernattaaolryPSfaetedhtoteleiosgssy 1LsnoadeborFaetsoireytony POJ RTERoomo110t aren, 1981 evort sumtetes ay: vE iznce=ntct a. GrarVGey,Eve6 eDomS3 e er viD cma2 raoLev(se{m= aionce isup, 000147 - TESTARTICLE SPECIFICATIONS : STUDY NUMBER: 3 TEST ARTICLE 1.0; __T-2942 CoC : TEST ARTICLE LOT HUMBER: __= DATE RECEIVED:___ 8/20/80 OATE STUDY Beans: H/27/80 DATE STUDY CowpLETED:_ 10/5/80 TEST ARTICLE STORAGE CONDITIONS: Room Temperature DILUENT(S)USED: prs TEST ARTICLE FORM: @s_ umn ___ soYte TEST ARTICLE QUANTITY RECIEVED: _ 10 grams SPECIAL HANDLING PRECAUTIONS: None HAZARD RATING: _ ommow 1 emus 1 4 __ emowercaL 1 4 LDgy ORAL ACUTE:__ 540 mg/kg SOLUBILITY OF TEST MATERIAL: (H)0; CHyOH; Lipid, etc.) _Hy0i CH 04; Acetone : 00148 Quality Assurance Statement The GSH 107-172 cell transformation assay 1s a highly reproducible method requiring several internal controls. Cells, media and serum are porting aersiand $07 Sr FISHES ar CUTIRIIONtS feds be fete poration into the test systen. This function has been carried aut by He. Nelson ond reviewed by Or. Garry in this initial study. The rav data and the Final report has been reviewed by our Quality Control Officer, We. Robert Kreiger. - ZA Vincent F. Garry, M07 Study Director Fh oS Soden ZZ Rabert. freiger (5. quality Control Officer 00149 . Executive Summary for Assays for both cytotoxicity and cell transformation have test article T-2942 CoC, using C3H 1011/2 cell cultures in been completed vitro. Cytotoxicity was cells. The date measured by a reduction of cloning showed an LDg, of approximately 50 efficiency for ug/ml. A dose treated range extending to 2 wide fron 0 to 200 ug/ml range of chemicals was tested reported in for the cytotoxic effect. Comparison literature indicates that the chenical studied (T-2942 CoC) exhibits a low order of cytotoxicity, utilizing C3 1071/2 cells. Transformation assays were conducted using the previously derived Lg, 2s 14 the day median colony dose, assay with a dose range spread for transformation and a of four log longer term units. Both the foci transformation regimen (38 day) were of transfornation was conpleted for the test article observed for either the colony T-2342 or foci CoC. Mo evidence assay methods using C3 107-172 cell cultures. : .00150_ : Introduction . This study was conducted for 3% by V. F. Garry, H.D., M.5., R. Nelson, B.A., R. Krieger, N.S. and M. Sinn, B.A., from 8/27/80 to 10/5/30 at the Environmental Pathology Laboratory of the University of Hinnesota, Minneapolis, Hinnesota. The experimental procedures employed are the test methods described by Reznikoff, Brankow and Heidelberger for the determination of transformation and cyto- toxicity in the C3 107-1/2 cell line. Aside from this classical methodology, we have provided 3 an early reporting feature to give a preliminary evaluation of results. All procedures are described in detail in the protocol included in the appendix of the report. 00151 - Materials and Methods Cen source Cryopreserved cells of the C3H 10T-1/2 clone 8 (Lot Number 3C1-1-4) utilizes oibntatihneesde sfrtoundieOrs. weC.reHdeeirdievlbeedrgferro,m Dpiarsescatgoer 11f.or of Sauthern California. The cryopreserved Tots BTahseicorRiegisneaalrchcelolf tshteockUnwiavsersity were prepared fron subcultures of the original cell line obtained at the Sth passage. Ce Culture conditions Stock containing cultures 108 heat for these studies inactivated fetal wbeorvienegroswenrusin (ERaeghelies BaCshaelm. MeCdoi.umLo(tSHE) eNfufmibceirenTc-y486a0n9d).tranPsrfioorrmattoiotnheefsftiucdiy,enctyheinserthuem pTortesweansce otfesdtierdecftorancdloniinndgirect abcatctienrgiaclarocrinomgyecnosp.laseIan cadodnittaimoinn,atitohne etontiirnseurceellthesyasdteeqnuawcasy tested of the for possible procedures. Test Cnemicats on empTlheoyeteestofch3a4aiCcoarlp,oraTt-i7o0n1.2 CCooCntwaazinerdaceiinveadcoonded8/2gl0a/s0s0 from vial uw.as HeacpCaprrmoic.k, mauattheolryiz1a0tmionofanwdhitienfpoorwmdaetri.on The material regarding in vas accompanied vivo toxicity. by a Totter. of The test chemical vas stored dissolved at in rspoeocmtrtaelmpegrraatduereO.HSO I(mFeidsihaetrelCyhembiecfaolreCou.se,LottheNumtbesetr materia was 702368) and dvaislutaedddedtotoapptrheopcruilattuerecsoncuenndterratsiuobndsu.ed Hithin yellow 20 minutes the Tight to avoid diluted chemies] photo-inactivatian. Ho more than 20 microliters of the diluted chenical vas added to the cultures to avoid solvent effects. The chemicals, Benzo(a)pyrene (Eastman Kodak Lot aHsumbpeorsit4i9v4e1) coanndtrodlis-.epoxybutane (Aldrich Chem. Co. Lot Number 082297) were used 00152 S Materials and Methods (continued Initia) Cytotoxicity peterination obtainPerdiorinttoheperffoormrmaonfcecytooftotxhiecittryanmsefsosrumraetnieonntsassaasy,expdroesseserdangbey data vas plating (cloning) efficiency. The test chemical T-2942 CoC was added to cultures seeded the day before at 300 cells per 60mm culture dish. After 24-hour exposure Tt-o28t6h2e CtoeCstwacshemriecmaolvsed iwnitShm] a BMcEonmpeldeitae cshuapnpgleemeonftmeeddiwai.th 1Th0e% tfeestatl chceaalifcaslerwuams; arpepplliiecdatethrcouultguhreas 4 plerogdosscealepoidnitluwteirones eraapnlgoiynegd afnrdoma 2t0o0tatlo o0.f1 siuxg/mdlo.se Spioxints were studied. The cultures vere refed with fresh media every three days. After a7nddaysstaiinnedcuwlittuhre,gietmhsea psltaatiens. werPeoswiatsihveedcoinntrPoBlSs,, fisxoeldvenwtithcoanbtsroollsu atenmdetshearnuoml saseline controls were included in this were counted and the plating efficiency study. The number of xas determined by the colonies formula: per plate PE = RAsvoeeorafdgeCeeMldoi.srCpoltaneas/tpleatse X 10 Transformation Assay dose The for approxinate fn vitro Lg, cytotoxicity dose was chosen as the the study of the transformation potential of the test chemical median t(rTa-n29s4f2ormCaoCt)i.on Tinhethteestcowlaosnycmoonddeuctweads aisnsetswsoedp.haseIsn. theIn stehcoendfirpsatrt,phafsocei, cterlalnsformation potential was determined. Phase 1 - Colony Transforation Potential Six replicate plates seeded with 300 cells per plate were treated with 200, 100, 50, 10, 1.0 and 0.1 ug/al to give appropriate two-fold and Tagarrithnic dose relationships for the test chemical T-2342 CoC. The positive con trol,benzola pyrene (5) vas tested at 10, 5, 2.5, 1.0, and 0.1 ug/l. A .asfotlevrent24cohnoturrosl ex(pDoMsSuOr)e,wastheinccullutduedres(1w0eurge/mir)e.fedAfftreesrh rmeemdoivaal anodf tthheerecahefmtiecralevery 3 days for 14 to 17 days. At 18 days after removal of the test chemical, the Plates were washed, fixed and stained as described before. Each calony was 00153 : carefully examined macroscopically and microscopically and scored for trans- formation according to criteria of Reznikoff, et al. Phase 2 - Faci Transformation Potential Remaining replicates at 6 replicates per dose were allowed to continue in culture for 38 days. Three dose increments were studied; 100, 10 and 1.0 ug/ml respectively. A positive control (butadiene epoxide) in dose 0.01, 0.001, 0.905 and 0.0001 ug/ml was included along with a increments of DISO solvent con- trol. The cultures were refed according to the schedule mentioned above for the first fourteen days. For the remainder of time in culture, the cells vere refed once weekly with DHE supplemented with 10% fetal calf serum. At 38 days after removal of the test chemical, the culture plates were processed as men- tioned above. Scoring for Transformation Focal areas of transformation Reznikoff as follows: are classified according to the criteria of Type I. Foct composed of monolayer cells are more densely packed than the background cells. This type is not considered malignant and is not scored. Type 11. Foci show messive piling up into virtually opaque multilayers. The cells are only moderately polar, thus criss-crossing is not pronounced. Fifty percent of Type II foci have been shown to be malignantly transformed. Tcyrpoesse1d11.arraFyosci oafredencsoemlpyosesdtaionfedhicgehlllys.polaEri,ghtfyi-bfriovbelapsteircc,entmulotfilTayypeerdI,II crfiocsis- have been shown to be malignantly transformed, Selected Type III foci tice, after which the will be retained from living cultures and cells will be cryopreserved for one year. subcultured If, during that tine, confirmatory in vivo tunorogenesfs studies are requested, these cells Will be made available for injection into a suitable host. 600154 J Materials and Methods, Continued Special C34 107-1/2 Early Reporting (Phase 1 Although the classic methodology for the C3H 10T-1/2 prescribed a 6-week course of study, recent data obtained by our Laboratory shows that the assay tine may be foreshortened to 15 to 20 days. Detailed microscopic study of the colonies allows for quantitation in this somewhat shorter frame using the classic morphologic criteria previously described. This early reporting capability is avaflable as preliminary information and is included in our report. Recording of Data and Reports All data from the cytotoxicity screening, transformation assay and early reporting inforaation are recorded in tabular form (see enclosure). A coded access number will be provided to allow retrieval of cryopreserved material, and the fixed and stained cultures. All reports will remain confidential, subject to disclosure by the Corporation. Although there are no specific FOA guidelines for in vitro carcinogenesis testing, we will in future, achere more closely to the Good Laboratory Practices protocol by formal establishment of : a quality control unit in our laboratory. References 1. Reznikoff, C., Brankow, D., Heidelberger, C. Cancer Research, 33:3231-3238, Dec. 1973. 2. Renzikoff, C., Brankow, D., Heidelberger, C. Cancer Research, 33:3239-3249, Dec, 1973. 3. Modal, s., Brankow, 0., Heidelberger, C. Cancer Research, 36:7, part 1, 2254-2260, July, 1976. 4. Bertram, J. S. Cancer Research, 17:51, 1976. 5. Bertram, J. S. Cancer Research, 37:2, 514-523, 1977. 6. Haber, 0. A., Fox, D. A., Dynan, W. S., Thilly, W. G., Cancer Research, 37:6, 1644-1648, June 1977. 7. Bertram, J. S., Cancer Letters 7:5, 288-298, September, 1579. 001SS Results . In plating the dose ranging cytotoxicity test, the chemical T-2%2 efficiency of 21.9% of control at 100 ug/al (Table 1). CoC showed To provide a an efficient description of the colony and foci were studied transformation potential of the chemical, both based on the initial cytotoxicity screening. In the colony mode, in log scale and eight dose points enclosing the in vitro LDgy two-fold dilutions, There was no morphologic were assessed evidence of transformation in the fourteen day colonies treated with the test chemical (Table 2) 100, Longer term 10, and 1.0 foci (38 days in culture) were studied in dose increments of ug/ml. Ho evidence of morphologic transformation were observed at any of the concentrations studied (Table 3). The positive control, benzo(a)pyrene frequency in the colony forming mode. In showed a the faci dose related transformation mode, the positive control (butadiene epoxide) transformants were again observed in studied. The combined number of type two and type three the dose increments foci varied fron . 43.0 to 21.3/plate. No abnormalities were detected in the solvent controls. 00156 conclusions The test chemical T-2942 Cot was studied fn C3H 107-172 cells for sorpho- logic evidence of transformation in vitro. At the dose levels employed, no evidence of transformation was observed. The positive controls, benzo(a)pyrene and dieporsbutane showed development of either Type 11-and Type TTT colonies or Type 11 or Type III foci. The solvent control showed rarsal morphology. 00157 .; ArrE1Tn1CoCBmLceoINmTEybR aACsonnToTPOLuTATnI1iN7G2nCFLETF8InCICeEERLELYSCoc 31 Corporation Crvent vr sary no. Tvestizter Tau coc Text Common 10. 75. Sta! ter TT Seoseon 200 va/bmo_s-e t9.a1ngewast _ vos | aahtEleicaaln|| bpirecenos or|| CTroalnosntioerssee ||T CTeoalnosntioersee(sTota Ii vg/m 6 Dishes/Dose | Controls Celless | Colonies Percent) | T2-842 cot . , |] || 10o0s Il)o snalses | || 2gl he n26ss 0.500 vous ore | Spoon 40.00 9.58 &| coma a0 . | i S. CONTROL* 40.8 99.27 i eu*1p0erucge/nmtl TrSaonlsvfeonrtmaCtoinotrnol(G(DoMSlO). = Aevrearaggee T Yo ThiPlatee eevtranstomation = H puerage toT . Thanean wvenp.E. percent of Contra) = priori F.E (Control) 00158 . Table 11 . COLOANFYT1ET4RRACNLEOSXWPFOEOSDRUMRCAEaTbITO0N10CM7HV1ED/M2ICCLAOcLLNIN8TG-8C5SE6F7FsICCoICSiCY 3M Corporation Cent V.F. Garry, up. Investigator T2992 coc Test Compound 1.0. Tumber _) Stuey Number oso solvent 200 use) - 1 vast Dose Range Dose Tovi. to"S.00 |2SelDiissyhoeiss/y| @] ee m ppeer.ce9nt)| pciorncternotlsof|| Combined Type Transformed 11 1 and Type 11 Colonies Transtarsed cotonies y Colantes/ce1(] (Total Catonies porsent wos [nose T2542 coc 0 o0 0 10 sl wm ze | sos | eas 2.6| ws | 799 Ew 30 1.0 | ner 81.40 ws | 12s o.22 HE i 2s | we | as ze [as | sss 54.21 note 10 | ao | ae | esas conrmon 430 | rem socom | 0 | war | ses To %0 | se | sem x0 2a 5 29.3 9.7 63.52 8.36 x 107 9.08 2.5 3.83 12.6 3 10 | 40s | ans 82.51 2.0 777107 12x07 6.16 5.32 | 01 | e433 | ze %.79 Z| contro se | sz - aa3x107 - 3.0 i - | sccomo | eso | res 53.65 - - | (3) 10 ug/m Sotvent Control (ps0) sverage No. T/plate (b) Percent Transformation (T/Col.Percent) = Average fio CoT./Plate {c) Transformation (T/Cell) = (@ 7. . percent = R AverageE %o. TT /Plate Average lo.of Cot/oish (6) 7. . percent of Control = nat FRR X 100 PE. of Dose FE (Tantra) 10 0159 . C3H 10T-1/2 CELLS | oe wo--Fomy Per Cell Plated 00160 Ag . C3 10T 1/2 CLOYE 8 TRANSFORMATION ASSAY 1T9h7e0'Cs34Re1z0n7ik1o/f2f,celBlankToiwneanids dHeeriidveeldberfgreorn!,m2ousuetileimzbercyoncilocnaclellcse.ils Infrotnhetehearly ifnoirtmiaatlionisaoslsaatye.foIenstbarbilefi,shtahne ianssavyitrcoonmsiesatssuroefoefxpcoasrucrienogoefnemsaimsm,alitahen cterlalnssctoultaivpaottienotniaolf nceawrlcyinosgeeendeidnCveiltlrosfofroraapperreicodiseofp3e0riotdo o35f dtaiymse.; fDoulrliownegdthbiysa otbitnaei,necdy.totBoexciacuistey atnhde mceolrlpsholaorgeimcorepvhiodelnocgeicoafllnyeohpolmaogsetniocustraanndsfosremnastiitiovneisto Tphoestncuoanvefrluoefncefociinhdiebtietcitoend, aabrneorpgraolporcteilolnuallartofoctihecadnoseeaosfilythebecdaertceicntoegde.n a7p-p1l0iedd.aysSoifmilcaurlltyu,retphreovniudmeber2 oqfuacnotliotnaiteisveofbasciesllsfordettehceteddetewrimtihninetitohne offirst cthyetotaobnxoircmiatly. celTlos coarnefirimnjetchteedcaricntionoga ensiucsceppottiebnltiealhosotf. theThetradnesvfeolrompemdentfoocif, mchaacrraocstceorpicoftuinncjresctewdi,thimnorp9h0oldoaygsicaclolnystiabtnuotremsalcocneflilrsm.ation of the neoplastic cTohleleaasgsuaeyshefsn tghoeodfrieelpdr.oducEiabchiliatsysayintoourbehapnedrsforamsedwelilnclausdeisn athopsoesitoifveourcontrol, asroelveunstedcotnotrcosltabalnidshfiveeachdosdeosepoipnotisnt.for the test chemical. Six separate cultures - 1. Procedures A. ge1l_source. fLroownpaDrs.sagHeeidcerlyboeprrgeesre.rvedFrocemlltsheofinitthiealC3cul1t0uTre1,/2aclToonwep8asswaegree ostbotcakined offirsctellssuspheansdebdeenine8stapbelricsehnetd.DMSOForplulsonmge-dtiearnansdtoraargee,pretsheervceedllsinare Tiinqauid-8n0itrocgeennt.igraFdoer sfrheoerzte-rt.ermPesrtioroadgiec,allvye,rsoaumtpilneelycumlatiurnetsainareceltlessted fionrhimbyitcioopnl.asmaIncotnhetampiansattisoinx,teecnlomnoinntghse,fficcloineinncgy aenfdficpioesntccoynfhlausenvcaeried ffroormmat1i2ont"o i1n6 apneyrceonft.ourTchuelrteuriessnoteesvtieddentcoedaotfe."spontaneous trans- 8. Cell Culture Conditions R10outpienrecleyn,t shteoactk-incauclttiuvraetsedarefetgarlownbovinineEagsleerumBaswai)thoMuetdiuthme(uBsEe)ofcoannttaii-ning obbitoatiicnse.d fTrhoem mceedritaifiiesdmacdoemmefrrcoimalposwoduerrceeds.formFetianl hicgahlfpusreirtuym wlaottserare : opnertihoidsicablasliys.testToedinfsourrethceoirntienfufietcyt oofn tcelsotniinngg seuffffiicciieenncty qanudantsietlieecsted . of scrun ore held on reserve. 00161 5 : C.. Test Chemicals CIhnefmoircmaaltsiontorebegartdeisntegdthsehouplhdysibcealidecnhtairfaicetderaisstipcrseci(seeg)lysaoslubpiolsistiyb,le, iTnachvivcohentiocxailcitryece(iLvDe5d0),{sancdodesdouracned adruepliicmaptoertarnetcorcdosnsoifdetrhaetitoensst procedure are maintained by the Laboratory. Cphreimaitcealssolvteontbe(esgt)udiDeNdSO.wilAlddbietitoensotfedtohne acheugm/imclalsbastioscuilntuanreapipsroaScucbosmepqluiesnhtedmedbiyamecdhfaangedsiluatrieoncsarruinedderoupthoutnodgerraphgiocld saffleutoryeslciegnhtt. light. 2. cytotoxicity PinriotrhetofoprerofforCmyatncoetooxficitthey mteraasnusrfeomremnttis.on aIsnsaayc,codrodsaencerawnigtehdatthae fpsrotoobctoalined established 1s measured by {n Rteerzmnsikoofff,plaBteirtnrgame,ffiBcriaennkcoyw,, imately 300/dish) are placed in culture and aAndfHiexieddelnbuemrbgeerrlo,f ccyetllostox(iacppirtoyxallowed to grow for 7 to 10 days. TPhheatepdercisenttahgeen doeftesurrmviinveidngUec)olotnhieesclodniivnigdedeffbyicitehnecyn.umbeRrouotifnecleyl,lschienniitciaallsly %sotanbedatredst2e4d-haoruer eadxdpeodsurtoe ptheeriocdu,ltutrheesch24emihcoaulrss aarfeterrempolvateidngw.ithAfatecrhanage of amreediaa.ppliTehde ctehlrlosugharea -relfoegd dforseeshsmceadlieauesviengry10thdroeesedapyosi.nts.TheThtreeset crheepmliiccaaltse LCualstculriense cpeorntrpoolfsntarsereienmcplluodyeded.withPosciatcihvestucdoyn.trolA,ll scohlevmeinctalconotrroslolvanedntsaedrduin- AHtiontshetoendtheofme7ditao a10redanyost,altlheowecdulttuoreesxceaerde 0f.i5xe%d,ofstthaienetdotaalndmetdhieanvuomlbuemre.of colonies are counted macroscopically and microscopically. 3. Transformation Assay : bThye Retzrnainksoffofr,matetioan lals.saayndismopdoirffioerdnedbyiBneratcrcaomr,d ewtitha1t.he methodology developed In line with this Cclualstsuircealdisahpepsroatcoh,acahpiperveoxismiaxterleypli3c0a0tecsellpserardeoseplpaotiendt,inonecaechtohfe dseisxirseedparate RCoonucteinnterlayta,iomninriannugne ohafsfboeurenddoseeterpmoiinnetds baryemesatsuudrieedmenitnsionfcrecalsoininnggdeiflfuitciioennscy. fDirloumtiaondsosewilwlhicbhe mcaaduesestwo5-0fotlod7o5rpmeorrcce,ntddeepcernedaisneg oinn ctlhoenisnlgopeeffoifcitehnecyC.yto- tidoexnitciictayl cutrovet.hatTheemplporyoecdedufroerscyftorotocxhiecmiictayl. additions to the cultures are . ASeTrumc.ultuRroeustinaerley,refreed-fweietdhingBasiasl doEnaegleesvemreyditahrweiethday1s0 monolayer of cells is acheived; usually 15 to 20 days. punetriclenta cfoetnafllucenatlf Thereafter, the Culturds are refed weekly. 00162 . 3 * 1 | -----Transformation Assay Continued ASttai35nedda,ysandafteexramirneemodvamlacroofsctohpeiccaolnlpyounadn,d maillcrocsuclotpuircealdliys,hesandarescofriexedd,for otnreanysefaorrmaatsioan.semAil-lpeormfantehnetfriexecdordandofsttaheinesdtudcyu.lturSecsorianrge orfetmaoirnpehdolfoogrical ettraanlslf.ormation fs in sccorance with the types described by Reznikoff, A. Scoring of Transformation ! oFfocaRlezanriekaosffloafstfroalnlsofwosr:mation are classified according to the criteria TTyhepeBaIc.kgrFooucnidcocmeplloss.ed Tohfismontoylpaeyeirs ncoetllscoanrseidmeorreed mdaelnisgenlayntpacakneddisthannot scored. TTHyepeCe11T.Ts aFroceionslhyowmomdaessriavteelypilpionlagrsupthiinstocvriirstsu-aclrloyssionpgaquies mnuolttiplraoynearusn.ced. Fifty percent of Type II foci have been shown to be malignantly transformed. TEyrpiess-1c11r.osseFdociarraareyscoofmpdoseendseloyf shtiagihnleydpoclealrl,s. fibEriogbhltays-tfiicv,e mpuelrtcielnatyeorfed, Type 111 foci have heen shown to be malignantiy transformed. cSuellteucrteedd tTwyipcee,I11aftfeorciwwhiilclh tbheerectelalisnedwilflrombe1cirvyionpgrecsuelrtvureeds faonrdonseub-year. rIef,qudesutreidn,g tthhaetsetciemel,lscwoinlflirmbeatmoarydeinavvaiivloablteunofroorgeinnejseicstiosntudiinetso 2aresuitable host. 4. Special C3 1011/2 Early Reporting cAolutrhsoeughofthsetucdyl,assrieccemntethdoatdaoloobgtyaifnoerdthbey oCu3rH 1L0aTbo1r/a2torpryesschroiwbsesthaat6t-hneeekassay tthienecmoalyonibees faolrloswhsortfeorncdquatonti15tattioon20idnaytsh.is Dseotmaeiwlheadt msihcorrotsecropfircamestuusdiyngof Cthaepabcilalsistiycimsorapvhaoilloagbilce acsritperreilaimipnraervyiouisnlfyordmeastciroinbeadn.d isThiisncelaurdleyd rienpoorutring report. 5. Recording of Data and Reports . rAeNporctaitang frionmfotrhmeaticoyntoatroexicrietcyordsecdreeinnintgab,ultarra,nsffoorrmma(tsieoeneansclsoasyuraen)d. earAlycoded aancdcestshe nufmibxeerd wainldl stbaeinperdovicdueldturteos.alloAlwl rerterpolrvtasl wiolfl crryeomapirnesecrovnefdidemnattiearli,al, 00163 PE 4 @i" . 2 ------Recording of Data and Reports, Continued " . sFLuaObbAojregacuttiodretylo.indPeirssaccltfoioscreuisrenPbvryoittortchooelcaCropcruiptnoorfagoterinwoeansr,ids btAyelsttthhioinsugg,hFewtdeherearaledhAeagrreeencntyoo. tshpeecGiofiocd 6. References : 1. R3e3z:n3i2k3o1f-3f2,36C,.,"DBerca.nk1o9u7,3.D., Hetdelberger, C. Cancer Research, 2. R3e3z:n3i2k3o9f-f3,249C,.,DBecr.ank1o9w7,3.0., Heidelberger, C. Cancer Research, 3. MPoanrdtal1,, 2S.2,84-B2r2a6n0k,ow,JulDy.,, H1e9i7d6.elberger, C. Cancer Research, 36:7, 4. Bertram, 0.5. Cancer Research, 17:51, 1976. 5. Bertram, J.S. Cancer Research, 37:2, 514-523, 1977. 6. H37a:b6e,r,1D6.4A4.-,164F8o,x,"Du.neA.,197D7y.nan, .5., Thilly, .G., Cancer Research, ~ : 7. Bertram, 3.5., Cancer Letters 7:5, 288-298, Septenber, 1575. 00164